The type of immune response generated to infection or other antigenic challenge can generally be distinguished by the subset of T helper (Th) cells involved in the response. The Th1 subset is responsible for classical cell-mediated functions such as delayed-type hypersensitivity and activation of cytotoxic T lymphocytes (CTLs), whereas the Th2 subset functions more effectively as a helper for B-cell activation. The type of immune response to an antigen is generally influenced by the cytokines produced by the cells responding to the antigen. Differences in the cytokines secreted by Th1 and Th2 cells are believed to reflect different biological functions of these two subsets. See, for example, Romagnani (2000) Ann. Allergy Asthma Immunol. 85:9-18.
The Th1 subset may be particularly suited to respond to viral infections, intracellular pathogens, and tumor cells because it secretes IL-2 and IFN-γ, which activate CTLs. The Th2 subset may be more suited to respond to free-living bacteria and helminthic parasites and may mediate allergic reactions, since IL-4 and IL-5 are known to induce IgE production and eosinophil activation, respectively. In general, Th1 and Th2 cells secrete distinct patterns of cytokines and so one type of response can moderate the activity of the other type of response. A shift in the Th1/Th2 balance can result in an allergic response, for example, or, alternatively, in an increased CTL response.
For many infectious diseases, such as tuberculosis and malaria, Th2-type responses are of little protective value against infection. Proposed vaccines using small peptides derived from the target antigen and other currently used antigenic agents that avoid use of potentially infective intact viral particles, do not always elicit the immune response necessary to achieve a therapeutic effect. The lack of a therapeutically effective human immunodeficiency virus (HIV) vaccine is an unfortunate example of this failure. Protein-based vaccines typically induce Th2-type immune responses, characterized by high titers of neutralizing antibodies but without significant cell-mediated immunity.
Moreover, some types of antibody responses are inappropriate in certain indications, most notably in allergy where an IgE antibody response can result in anaphylactic shock. Generally, allergic responses also involve Th2-type immune responses. Allergic responses, including those of allergic asthma, are characterized by an early phase response, which occurs within seconds to minutes of allergen exposure and is characterized by cellular degranulation, and a late phase response, which occurs 4 to 24 hours later and is characterized by infiltration of eosinophils into the site of allergen exposure. Specifically, during the early phase of the allergic response, allergen cross-links IgE antibodies on basophils and mast cells, which in turn triggers degranulation and the subsequent release of histamine and other mediators of inflammation from mast cells and basophils. During the late phase response, eosinophils infiltrate into the site of allergen exposure (where tissue damage and dysfunction result).
Antigen immunotherapy for allergic disorders involves the subcutaneous injection of small, but gradually increasing amounts, of antigen. Such immunization treatments present the risk of inducing IgE-mediated anaphylaxis and do not efficiently address the cytokine-mediated events of the allergic late phase response. Thus far, this approach has yielded only limited success.
Administration of certain DNA sequences, generally known as immunostimulatory sequences or “ISS,” induces an immune response with a Th1-type bias as indicated by secretion of Th1-associated cytokines. Administration of an immunostimulatory polynucleotide with an antigen results in a Th1-type immune response to the administered antigen. Roman et al. (1997) Nature Med. 3:849-854. For example, mice injected intradermally with Escherichia coli (E. coli) β-galactosidase (β-Gal) in saline or in the adjuvant alum responded by producing specific IgG1 and IgE antibodies, and CD4+ cells that secreted IL-4 and IL-5, but not IFN-γ, demonstrating that the T cells were predominantly of the Th2 subset. However, mice injected intradermally (or with a tyne skin scratch applicator) with plasmid DNA (in saline) encoding β-Gal and containing an ISS responded by producing IgG2a antibodies and CD4+ cells that secreted IFN-γ, but not IL-4 and IL-5, demonstrating that the T cells were predominantly of the Th1 subset. Moreover, specific IgE production by the plasmid DNA-injected mice was reduced 66-75%. Raz et al. (1996) Proc. Natl. Acad. Sci. USA 93:5141-5145. In general, the response to naked DNA immunization is characterized by production of IL-2, TNFα and IFN-γ by antigen-stimulated CD4+ T cells, which is indicative of a Th1-type response. This is particularly important in treatment of allergy and asthma as shown by the decreased IgE production. The ability of immunostimulatory polynucleotides to stimulate a Th1-type immune response has been demonstrated with bacterial antigens, viral antigens and with allergens (see, for example, WO 98/55495). Interestingly, ISS act through a cell surface receptor (the Toll-like receptor 9, or TLR9), although one recent report suggests that internalization of ISS is necessary for activation of TLR9. Chuang et al., 2002, J. Leukoc. Biol. 71(3):538-44; Ahmad-Nejad et al., 2002, Eur. J. Immunol. 32(7):1958-68.
Other DNA-based therapies must be internalized before they can act. While certain tissues can take up limited amounts of nucleic acids (Wolff, 1997, Neuromuscul. Disord. 7(5):314-8), much attention has been focused on formulations to improve internalization of gene therapy and antisense constructs. Such formulations typically involve the use of a cation to condense the DNA and one or more lipids to (frequently in the form of liposomes) to promote membrane fusion. For example, Semple et al., 2001, Bioch. Biophys. Acta 1510:152-166, Cappaccioli et al., 1993, Bioch. Biophys Res. Comm. 197(2):818-25, Maurer et al., 2001, Biophys. J. 80:2310-26, Shi et al., 2001, Nucl. Acid Res. 29(10):2079-87, and Meyer et al., 1998, J. Biol. Chem. 273(25):15621-27, all disclose the use of cationic lipid/DNA compositions in serum-containing media in vitro. Other cation/DNA combinations have been tested in serum-containing medium, including DNA with poly-L-lysine (Ginobbi et al., 1997, Anticancer Res. 17:29-36, Stewart et al., 199, Mol. Pharmacol. 50:1487-94, and Gonzalez-Ferreiro et al., 2001, J. Controlled Release 73:381-90), cationic peptides (Junghans et al., 2000, Nucl. Acid Res. 28(10):e45, Wyman et al., 1997, Biochemistry 36:3008-17), polymerized cationic detergents (Dauty et al., 2001, J. Am. Chem. Soc. 123(38):9227-34)polyaminolipids (Guy-Caffey et al., 1995, J. Biol. Chem. 270(52):31391-96), cationic block copolymers (Kabanov et al., 1995, Bioconj. Chem. 6(6):639-43), and cationic dendrimers (Bielinska et al., 1996, Nucl. Acid Res. 24(11):2176-82, Yoo et al., 1999, Pharmaceut. Res. 16(12):1799-1804). Legendre et al., 1993, Proc. Nat'l Acad. Sci. USA 90:893-97, discloses the use of several different cations, including the cationic antibiotics gramidin S and polymyxin B, to improved transduction of gene therapy constructs. U.S. Pat. No. 5,744,166 discloses pharmaceutical compositions of a polycationic polymer and DNA that may optionally contain poloxamer 407 or gelatin. Chavany et al. (1992, Pharmaceut. Res. 9(4):441-49) discloses the effects of a nonionic detergent (poloxamer 188) on a formulation including an alkyltrimethylammonium salt (CTAB) and DNA. Additionally, International Patent Application No. WO 01/15726 discloses combinations of charged lipids and ISS.